Bivalent non-human gal-α1-3-gal glycan epitopes in the Fc region of a monoclonal antibody model can be recognized by anti-Gal-α1-3-Gal IgE antibodies

Figures & data

Figure 1. Overall synthetic scheme for a monoclonal antibody comprising various α1,3-galactosylated glycans in the Fc region. MBD-α1–3-GalTase: Maltose-binding domain-conjugated α1–3-galactosyltransferase; UDP-Gal: uridine diphosphate galactose.

Glycan structures are represented by connected squares and circles, and reagents showing the synthesis of various α-Gal-containing glycans on the top. The bottom shows a schematic representation of the removal of glycans from a cartoon representation of a monoclonal antibody, followed by incorporation of the above glycans.

Figure 2. Synthesis of α1–3-Gal-containing mono-β-galactosylated (G1T) Glycans. (a, b) Mono-β-galactosylated glycans (1) and (2) were reacted with α1–3 galactosyltransferase (MBD-α1–3-GalTase) to produce mono-α/β-galactosylated glycans (4) and (5). Overlaid HPAEC-PAD chromatograms show the starting materials and products. (c) HPAEC-PAD traces showing the purified α1–3-Gal-containing glycans (4 and 5) following purification by Hypercarb PGC-HPLC. (d, e) Mono-α-galactosylated (4) and (5) were partially degraded by α1–3,6-galactosidase (black traces) to produce compounds (1) and (2). As a negative control, starting materials were also reacted with β-1,4-galactosidase (red traces), which showed no reactions as expected.

Five sets of chromatograms showing the change in locations of the peaks along the x-axes, which shows time in minutes, increasing from left to right. Above the first, second, fourth, and fifth chromatograms, a synthetic scheme depicting the transformation of glycan structures represented by connected squares and circles is shown. Treatment of the starting glycan structures on the left of the arrows with reagents stated above the arrows results in formation of new glycan structures to the right of the arrows and is represented by changes in the location of peak heights along the x-axes below.

Figure 3. Synthesis of α1–3-Gal-containing bi-β-galactosylated (G2T) glycans. (a) Synthetic scheme for reaction of bi-β-galactosylated glycan (3) with α1–3-GalTase to produce mono- and bi-α-galactosylated glycans (6-8). Overlaid (b) HPAEC-PAD and (C) PGC-HPLC chromatographs show the starting material (blue trace), and partial reactions at 37°C after 2 and 8 h leading to the formation of α-galactosylated glycans (6-8). PGC-HPLC chromatographs show the presence of β- and α-anomers for each glycan. (d, e) Treatment of purified mono-α-galactosylated glycan (d) (6) and (e) (8) with α1–3,6 galactosidase (black traces) confirms the presence of an α1–3-Gal epitope. (f, g) Reaction of mono-α-galactosylated glycans (f) (6) and (g) (8) with LacZ β1–4-galactosidase (red traces) results in the formation of compounds (5) and (4), respectively. The characteristic re-arrangement byproduct of the LacZ β1–4 galactosidase reaction is denoted by an asterisk. As a reference, G1(α3)T (4) and G1(α6)T (5) glycans synthesized independently (i.e., from Figure 2a, b) are shown. (h) Synthesis of mono-α-galactosylated glycan (8) using mono-α/β-galactosylated glycan (4), UDP-Gal and β1–4-galactosyltransferase. HPAEC-PAD trace of the final product is shown. (i) Bi-α-galactosylated (7) was partially cleaved with α1–3,6-galactosidase to produce compounds (3, 6, and 8, black trace). As a negative control, glycan (7) was also reacted with LacZ β-1,4-galactosidase, which showed no reactions (red trace).

Eight sets of chromatograms arranged in two rows and four columns, with synthetic schemes depicting the transformation of glycan structures represented by connected squares and circles shown above them. Treatment of the starting glycan structures on the left of the arrows with reagents stated above the arrows results in formation of new glycan structures to the right of the arrows. The chromatograms below show the corresponding peaks representing the starting glycans and the reacted products, with some showing additional peaks of a known glycan structure as a reference. The x-axes show time in minutes, increasing from left to right.

Figure 4. Characterization of palivizumab transglycosylated with α1–3-Gal-containing Fc N-glycans. (a, b) SDS-PAGE gels of palivizumab transglycosylated with (a) conventional human-type glycans and (b) non-human α-galactosylated glycans, and the deglycosylated starting material (S.M.). Lanes with both product and starting materials are shown to confirm differences in band migration. A protein ladder is shown on the first lane of each gel. (c, d) Analysis of transglycosylated glycans, which were cleaved from the palivizumab protein backbone with PNGase F, and analyzed by (c) HPAEC-PAD, and (d) CE-LIF following APTS Labelling. Glycans cleaved from commercial palivizumab is shown as a reference.

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Two SDS-PAGE gels on the top showing various dark bands at different heights. Along each column, single bands show that the monoclonal antibody was either completely glycosylated or deglycosylated, as shown by higher or lower bands, respectively. Columns with double bands are a combination of glycosylated and deglycosylated bands to show their relative sizes to each other. Below are two boxes with each showing a set of chromatograms showing the purity glycans of the remodelled monoclonal antibodies. At the bottom of each box, the second chromatographic trace from the bottom contains numerous peaks and is a glycan mixture from a commercial product. Above these are traces showing individual peaks representing single glycans that were incorporated into the monoclonal antibodies. On the left, some show small amounts of extra peaks, which align with the buffer blank peaks shown at the bottom trace.

Figure 5. Homogeneous bi-α-galactosylated glycoforms in the Fc region binds to different commercially available anti-α1,3-Gal IgE antibodies by ELISA. Binding to (a) clone 16D9 and (b) clone 10H8 anti-α1,3-Gal IgE antibodies. Bi-α-galactosylated glycans (purple diamond data points) bind relatively higher than mono-α-galactosylated Fc glycans. Commercially available cetuximab containing 27–34% Fab bi-α-galactosylated glycans are shown as a comparison. Commercially available palivizumab (Synagis) and deglycosylated palivizumab are shown as negative controls (gray data points). Error bars represent mean ± standard deviation from three separate experiments.

Two graphs with different colored curves in each. The x-axes are monoclonal antibody concentrations increasing from left to right in micrograms per milliliter, while the y-axes have normalized absorbance values increasing from bottom to top. The red lines and triangles represent the commercial cetuximab control, and the purple lines and diamonds represent the bi-α-galactosylated Fc glycans. The purple lines and data points are more similar to the red lines and points in the left graph than the right graph. For both graphs, the red and purple lines and shapes are higher along the y-axes than the other colored lines and shapes.

Figure 6. α1–3- or β1–4-galactosylation on each α3 or α6 antenna differentially affects FcγRIIIA binding. (a) Absence of β1–4-galactosylation (Gal-β1–4-GlcNAc) on the α6 antenna decreases binding (G1(3)F, yellow triangle) compared to its presence (G1(6)F and G2F, green and blue triangles, respectively). (b) α1–3-galactosylation (addition of α1–3-Gal epitope) on the α3 antenna increases FcγRIIIA binding (orange circles) compared to parental G1(3)F (yellow triangles), with additional β-galactosylation of the α6 antenna further increasing binding (yellow squares). However, addition of second α1–3-Gal epitope on the α6 antenna decreases binding (purple diamonds). (c) α1–3-galactosylation on the α6 antenna decreases binding regardless of substitution at the α3 antenna (cyan circles vs green squares vs purple diamonds) compared to parental G1(6)F and G2F (green and blue triangles, respectively). Error bars represent mean ± standard deviation from three separate experiments.

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Three graphs with different colored curves in each. The x-axes are monoclonal antibody concentrations increasing from left to right in micrograms per milliliter, and the y-axes are the normalized absorbance values increasing from bottom to top. In the left graph, the green and blue lines and triangles overlap and are higher in the y-axes values than the yellow curve and triangles. The middle graph shows five different colored lines and data points at different positions. The left-most curve is a dark yellow line with square data points. The right-most curve is another dark yellow line, but with triangle data points. Purple, blue, and orange lines and data points lie between these two dark yellow curves. The right graph shows another set of five different colored lines and data points at different positions. The left-most curves are overlapped green and blue lines with triangle data points. The right-most curve is a turquoise line with circle datapoints. A dark green and purple line and datapoints are between the left-most and right-most curves.